PANoptosis signaling enables broad immune response in psoriasis: From pathogenesis to new therapeutic strategies

Background Accumulating evidence suggests that regulated cell death, such as pyroptosis, apoptosis, and necroptosis, is deeply involved in the pathogenesis of psoriasis. As a newly recognized form of systematic cell death, PANoptosis is involved in a variety of inflammatory disorders through amplifying inflammatory and immune cascades, but its role in psoriasis remains elusive. Objectives To reveal the role of PANoptosis in psoriasis for a potential therapeutic strategy. Methods Multitranscriptomic analysis and experimental validation were used to identify PANoptosis signaling in psoriasis. RNA-seq and scRNA-seq analyses were performed to establish a PANoptosis-mediated immune response in psoriasis, which revealed hub genes through WGCNA and predicted disulfiram as a potential drug. The effect and mechanism of disulfiram were verified in imiquimod (IMQ)-induced psoriasis. Results Here, we found a highlighted PANoptosis signature in psoriasis patients through multitranscriptomic analysis and experimental validation. Based on this, two distinct PANoptosis patterns (non/high) were identified, which were the options for clinical classification. The high-PANoptosis-related group had a higher response rate to immune cell infiltration (such as M1 macrophages and keratinocytes). Subsequently, WGCNA showed the hub genes (e.g., S100A12, CYCS, NOD2, STAT1, HSPA4, AIM2, MAPK7), which were significantly associated with clinical phenotype, PANoptosis signature, and identified immune response in psoriasis. Finally, we explored disulfiram (DSF) as a candidate drug for psoriasis through network pharmacology, which ameliorated IMQ-mediated psoriatic symptoms through antipyroptosis-mediated inflammation and enhanced apoptotic progression. By analyzing the specific ligand—receptor interaction pairs within and between cell lineages, we speculated that DSF might exert its effects by targeting keratinocytes directly or targeting M1 macrophages to downregulate the proliferation of keratinocytes. Conclusions PANoptosis with its mediated immune cell infiltration provides a roadmap for research on the pathogenesis and therapeutic strategies of psoriasis.


Introduction
Psoriasis is considered a genetic and immune-mediated inflammatory disorder, manifesting in patient skin or joints or both [1].The prevalence of psoriasis among people peaks as high as 11.43 % but varies according to region, presenting at any age with a marked negative  S2. (B) Volcano plots highlighting the significantly different PANoptosis-related key genes (P < 0.05) between lesional and nonlesional skin from psoriasis patients shown in Table S3.Red  effect for individuals and society [2].Psoriasis is associated with a high risk for systemic comorbidities, including psoriatic arthritis, cardiometabolic syndrome and various psychiatric disorders, that may influence morbidity or curative effects among these patients [3].
Thus far, the complex causes and pathogenesis of psoriasis have not yet been fully elucidated.Over the past two decades, previous findings have highlighted the inflammatory response and causal immunological circuits as the key drivers of psoriasis pathogenesis, reflected by dysregulated proliferation and aberrant differentiation of keratinocytes and excessive immune cell infiltration [1,4].These include many classic (CD4 + and CD8 + T cells, dendritic cells (DCs), macrophages and neutrophils) and nonclassic immune cells (keratinocytes) [3,4].In psoriasis, epidermal cells seem more susceptible to external harmful triggers, leading to cell damage or even cell death.
In the present study, we provided a comprehensive report characterizing the expression profile and potential role of PANoptosis in psoriasis.Based on the PANoptosis signature, we identified two distinct PANoptosis patterns, which showed a significantly increasing level of immune cell infiltration in high PANoptosis-related patterns, such as keratinocytes and macrophages.Subsequently, WGCNA integrated with the Enricher database identified disulfiram (DSF) as a potential drug for psoriasis therapy, and its therapeutic role was verified in psoriasis in vivo.In summary, our results highlighted PANoptosis signaling within its mediated immune cell infiltration involved in the pathogenesis of psoriasis, which provided a breakthrough into the therapies and improved the outcomes of patients with psoriasis.

Human samples
Skin biopsies were obtained from the Department of Dermatology in Xiangya Hospital, Central South University (CSU).We enrolled 8 patients with psoriasis and 6 age-matched healthy individuals, who signed informed consent.All of the processes in human sample usage or the experiment were approved by the ethical committee of the Xiangya Hospital of CSU, following the principles of the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report (IRB number 201611610).
The mice were randomized into four groups and acclimatized for 7 days before being shaved on the back.In the imiquimod (IMQ) group, they were received locally with a daily topical dose of 42 mg IMQ (5 %) cream (Mingxin Pharmaceuticals, Sichuan, China) for 7 consecutive days [19].Meanwhile, 42 mg Vaseline jelly was used as a control for the daily topical treatment with IMQ.Disulfiram (DSF, Cat#: S1680, Sellec, Houston, MS, USA) was dissolved in 10 % dimethyl sulfoxide (DMSO, Sigma, St, Louis, MO, USA) at 0.1 M and 0.01 M in 50 μL and utilized in two drug-treated groups every other day.Meanwhile, 10 % DMSO was utilized as a control for the every-other-day administration of DSF (Fig. 4A).Psoriasis Area and Severity Index (PASI) scores were assessed for each experimental mouse each day by two independent laboratory assistants [20,21].On Day 8, the body weight was tested, and spleen and skin samples were taken from the sacrificed mice.

Data source and analysis for PANoptosis
The terms 'Psoriasis' and 'Homo sapiens' were used to search in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) with no limits on publication time.Exclusion criteria: 1) therapy intervention; 2) full-data not available; and 3) duplicate data.Inclusion criteria: 1) RNA sequencing (RNA-seq) data or single-cell sequencing data (scRNA-seq) of psoriasis; 2) the sample obtained from nonlesion and lesional skin of psoriasis (aiming to reduce individual differences); and 3) at least 20 individuals involved in the study.
We selected the RNA-seq data (ID: GSE30999; platform: GLP570) as the training set in our analysis, which had a large sample size, and the participants were in the same pathological stage.GSE30999 consists of 85 moderate-to-severe psoriasis and 85 matched biopsies of nonlesional skin.Furthermore, we selected another dataset from the same platform (GLP570) (GSE13355, GSE14905, GSE34248, GSE41662, GSE41664, GSE78023, and GSE117239) as a validation set for verifying the PANoptosis-related subtyping and marker gene levels.A systematic assessment of the psoriasis transcriptome is shown in Fig. S1, and the information of the involved dataset is shown in Table S1.Under the R environment, the Robust Multiarray Average (RMA) algorithm in the "Affy" package was performed to preprocess the raw data.After background correction, quantile normalization, probe summarization, and eliminating the unpaired data, the normalized dataset was used for the follow-up study.

Differentially expressed gene (DEG) analysis and functional enrichment analysis for the PANoptosis signature
The normalized expression data of PANoptosis-related genes were analyzed using the "limma" and "ggplot2" packages for DEG identification (P values < 0.05 were considered statistically significant).The activity score of the PANoptosis signature was determined through gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) using the "gsva" package.

Classification of psoriasis based on the PANoptosis signature
Unsupervised cluster analysis was carried out to identify the distinct PANoptosis-(k = 2)/pyroptosis-(k = 3)/apoptosis-(k = 2)/necroptosis-(k = 2)-related patterns in psoriasis by increasing the clustering variable (k) from 2 to 9 and selecting as the most appropriate one using the R package "ConsensuClusterPlus" [22].Meanwhile, a Sankey chart was used to visualize the association between molecular characteristics and clinical features using the R package "ggalluvial" [23].Furthermore, the classification was validated by principal component analysis (PCA) using the "prcomp" function in the R package "stats" [24].

Analysis of functional enrichment pathways, which highlights immune signatures
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out using the R package ("clusterProfiler" [25], "enrichplot" and "ggplot2″) to reveal the molecular characters in PANoptosis-high-related patterns.Xcell and single-cell analyses were performed to calculate the levels of infiltrating immune and stromal cells using the R package [26].

Hub gene screening for the prediction of candidate drugs
Weighted gene coexpression network analysis for specific modules was performed using the "WGCNA" R package (threshold value = 0.9, power (β) = 6).The most relevant module genes (|Gene Significance| (| GS=) > 0.2 and |Module Membership| (|MM=) > 0.8) were selected as the hub genes for the prediction of candidate drugs [27].Both these selected hub genes and the top 100 DEGs in the PANoptosis-based pattern were submitted to the Enricher database.These candidate drugs were then scored through the structure-based virtual screening of ZBP1 (Z-DNA binding protein 1, a key trigger in PANoptosis signaling).Targeted to high-scoring candidate drugs, DSF was identified as a candidate drug for psoriasis.The top 100 DEGs were submitted to the connectivity map (Cmap) database to validate DSF findings.To further analyze the drug-involved mechanism, irGSEA was performed to score the DSF enrichment of each immune cell.The CellChat package was used to analyze the enriched cellular communication.

Hematoxylin and eosin (H&E) staining assay
Skin Sections (3-5 µm) were deparaffinized and washed as described in the IHC analysis.First, these sections were stained with hematoxylin for 2 min, separated with 1 % hydrochloric acid-ethanol solution for 5 s and washed with PBS for 3 × 5 min.Then, these sections were stained with eosin for 3 min and dehydrated using 70 % ethanol for 10 s, 85 % ethanol for 20 s, 95 % ethanol for 1 min and 100 % ethanol for 1 min in sequence.Neutral gum mounting medium was added to the dehydrated sections for visualization.

EthD III staining assay
Pyroptosis and necroptosis could be assessed by staining with EthD-III in red-positive cells (Biotium, Fremont, CA, USA) [29,30].The skin sections were incubated with EthD III (10 μg/mL) for 10 min at RT and washed with PBS for 3 × 5 min.Afterward, these sections were co-stained with Hoechst (Sigma, St. Louis, MO, USA) for 5 min and washed with PBS for 3 × 5 min for visualization.

TUNEL staining assay
Skin sections were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (Meilunbio, Dalian, China), which marked apoptotic cell death in the red positive cells [31].Afterward, these sections were co-stained with Hoechst (Sigma, St. Louis, MO, USA) for 5 min and washed with PBS for 3 × 5 min for visualization.Apoptotic rate (%) = the number of red-positive cells/the total number of cells (same fields).

Statistical analyses
Statistical analysis was conducted using Prism-pro-6.0and R package.The quantitative data from the immunohistochemistry analysis are shown as the medians.One-way analysis of variance (ANOVA), Student's t test, Mann-Whitney test and Kruskal-Wallis test were used in this study (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS: not significant).

PANoptosis signaling highlighted in psoriasis
A total of 127 PANoptosis-related genes were involved in the training set (ID: GSE30999; platform: GLP570) (Fig. 1 A and Table S2), among which 86 genes were statistically significant in the psoriatic group (P < 0.05) and 60 genes were significantly upregulated (such as GZMB, CASP1, CASP4, AIM2, MYD88, CYCS, MLKL, IL1B, GSDMC, GSDME, PIK3R2, BAX, BAK1, FADD, RIPK3, NLRP3, GSDMD, IL1A, BAD, and ZBP1) (Fig. 1B and G, and Table S3).Moreover, GSEA systematically showed highly upregulated PANoptotic signaling in psoriasis (Fig. 1C).For further evaluation, GSVA analysis was performed, which also suggested significantly activated PANoptosis in psoriasis (Fig. 1D and F).In a previous study, necroptotic and apoptotic cell death was reported in skin lesions from patients with psoriasis [17,32].Additionally, we analyzed the expression of the critical numbers involved in PANoptosis (pyroptosis) signaling in skin lesions of psoriatic patients.The gene expression of these key members (Caspase-1, Nlrp3, Gsdmd, Il-1β) was significantly increased in the lesions of psoriasis (P < 0.01) compared with nonlesional skin (Fig. 1E).Additionally, the protein levels of CASP1, NLRP3, GSDMD and IL-1β in the epidermis were also significantly increased in the skin lesions of psoriatic patients (Fig. 1H and I).The isotype control and the negative control revealed a high specificity of the primary antibody used in human samples (Fig. S2).Meanwhile, a significantly activated PANoptosis signature was also identified in psoriasis in the validation sets, combined with the increased level of key genes (Fig. S3).Overall, PANoptosis-related signaling was highly activated in psoriatic lesions.

Classification and characterization of PANoptosis patterns
Based on the highlighted PANoptosis signaling, a consensus clustering analysis was applied, which identified two distinct patterns, termed the unrelated PANoptosis pattern (PAN_cluster_A) and the highly related PANoptosis pattern (PAN_cluster_B) (Fig. 2A).PANoptosis is a simultaneous single-cell-induced RCD that cannot be accounted for by any of these three involved RCDs alone [33].Therefore, we compared the PANoptotic patterns with the separate RCD-based patterns.In the Sankey chart, PANoptotic patterns make the classification more ergonomic on the molecular characters and the clinical phenotype in psoriasis (Fig. S4).Subsequently, PCA was performed to validate the PANoptosis-based classification (Fig. S5A and B).In other validation sets, PANoptosis-based classification could also effectively distinguish psoriasis lesions from nonlesional skin (Fig. S6).
To further reveal the mechanism, multiple variation analysis and functional enrichment analysis were used.The top 100 DEGs involved in the high-PANoptosis-related pattern were selected for subsequent drug prediction (Fig. 2B and C).In the high-PANoptosis-related pattern, a variety of inflammatory and immune-related factors combined with their pathways were highly enriched (Fig. S5C-E).Consistently, GSEA and GSVA also revealed significantly enriched inflammatory and immune pathways in high-PANoptosis-related patterns, such as IL-17, NF-κB, NOD-like receptor, TNF, Toll-like receptor, and Th1/2 cell differentiation signaling pathways (Fig. 2D and Fig. S5C).Targeting the immune signaling pathways, we further analyzed the landscape of immune cell infiltration.Combined X-cell and single immune analysis, the infiltrating immune cells were significantly increased in high-PANoptosisrelated patterns, such as keratinocytes, macrophages, and M1 macrophages (Fig. 2E, Fig. S7 and Fig. S8).This result suggests that the PANoptosis reaction is closely associated with the immune response during the pathogenesis of psoriasis.

Identification of the hub gene modules for drug prediction based on PANoptosis-mediated immune signaling involved in psoriasis
Based on 20,858 DEGs, the distinct gene modules in PANoptosismediated immune signaling involved in psoriasis were identified using WGCNA (Fig. 3A, Fig. S9A and S9B).Among these gene modules, the black module was positively and significantly correlated with both the psoriatic phenotype and PANoptosis signaling (R > 0.5 and P < 0.05 shown in Fig. 3A).Subsequently, 471 hub genes were screened as the module hub genes based on GS > 0.2 and MM > 0.8 (Fig. 3B).Among these, 234 hub genes were merged with upregulated DEGs, which served as candidates for drug prediction through the Enricher database (Fig. S9C).Based on the DEGs and hub genes, candidate drugs were identified (the top 200 shown in Tables S4 and S5).Among these, 75 overlapping drugs and 37 FDA-proven drugs are shown in Table S6.Then, the 37 FDA-proven drugs were scored through structure-based virtual screening of ZBP1 (a key trigger in PANoptosis signaling [34]), as shown in Table S7.For the high-scoring candidate drugs, the top 10 candidate drugs were collected as the candidate drugs for psoriasis, as shown in Fig. 3C.Birinapant (TL32711) (ranked 1st) and DSF (ranked 2nd) are approved by the FDA [35].Birinapant, an antagonist of apoptosis proteins (IAPs), has been regarded as a therapeutic strategy with chemotherapeutics in advanced or metastatic solid tumors and hematological cancers [36,37].DSF is an antagonist of pyroptosis proteins (GSDMD) and an inducer of the apoptotic pathway, which is closely related to a wide range of biological activities involved in various inflammatory diseases, such as the inflammatory response, cellular death progression, and aldehyde dehydrogenase metabolism [38][39][40][41].A previous study reported that DSF could attenuate atopic dermatitis-like skin lesions [42] and topical infections [43].Thus, in this study, we selected DSF as a candidate drug to attenuate psoriatic lesions.
To validate the DSF findings, the top 100 DEGs were submitted to the CMap database, which showed that the connectivity score of DSF was 0.4249 (Table S8).Furthermore, we performed molecular docking to identify the possible binding between DSF and ZBP1 (docking score, − 6.56) (Fig. 3D).The molecular docking also identified a high docking score between DSF and Caspase-1 (one of the hub genes involved in the PANoptosis-related signature) (docking score, − 8.673), as well as CXCL1 (one of the hub genes not involved) (docking score, − 1.806) (Fig. 3D).This result indicated that DSF might target the PANoptosis pathway in psoriasis.

DSF ameliorates IMQ-mediated psoriatic lesions
To investigate the effects of DSF on psoriasis, we induced a psoriatic mouse model with topical IMQ cream (Fig. 4A).Our data showed that DSF could significantly improve psoriasis-like symptoms.In detail, the score evaluation of erythema, induration, and desquamation of the plaques was reduced in psoriasis-like mice treated with DSF (Fig. 4B).Moreover, histological analysis showed that DSF decreased the thickness of the skin-fold and spleen index in the psoriatic mouse group (Fig. 4C-F).Fig. 2. A novel molecular pattern was established based on panoptosis-related characteristics.(A) Two distinct Panoptosis-related subtypes were eventually identified using unsupervised clustering.Unsupervised clustering analysis was performed by increasing the clustering variable (k) from 2 to 9, and k = 2 was selected as the most appropriate one through the R package "ConsensuClusterPlus".(B) Heatmap of the top 30 significantly differentially expressed genes in the two clusters.(PAN_cluster A: unrelated PANoptosis group; PAN_cluster B: highly related PANoptosis group).(C) Volcano plots highlight the significantly different genes between PAN_cluster A and PAN_cluster B (P < 0.05 and |log FC= > 1).(D) GSEA highlighted the significant reaction pathways in psoriasis, especially the immune response pathway.(E) The heatmap shows the normalized scores of increased immune cell infiltration using Xcell analysis in PAN_cluster A (unrelated PANoptosis group) and PAN_cluster B (highly related PANoptosis group).Purple represents cells with lower infiltration, and red represents cells with higher infiltration.Statistical differences were compared by the t test.P < 0.05 was regarded as statistically significant.The RNA-Seq data were obtained from the GEO database (GSE30999), which involved 85 nonlesional and 85 lesions of psoriasis.RNA-Seq, RNA sequencing.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) X.-m.Hu et al.In a previous study, DSF served as a pyroptotic inhibitor by blocking GSDMD pore formation, contributing to counteracting inflammation [41].It has also been reported that DSF enhances apoptosis progression via the ROS/MAPK pathway [44].Therefore, we hypothesized that DSF might ameliorate IMQ-mediated psoriatic lesions through antipyroptotic processes and enhanced apoptotic processes.Consistently, EthD-III staining experiments showed that the number of EthD-III-positive cells was reduced in the epidermis of the therapeutic psoriatic lesions (Fig. 4G and I).TUNEL staining also showed that enhanced apoptotic cell death occurred in the high-concentration DSF group (Fig. 4H and J).
Furthermore, we detected the expression of critical proteins involved in pyroptosis/apoptosis in mouse lesions.Compared to the control group treated with Vaseline, the expression of NLRP3, Caspase-1, GSDMD, and IL-1β in the epidermis was significantly increased in IMQ-induced psoriatic-like skin lesions (Fig. 5A and B).After treatment with DSF, the expression of NLRP3, Caspase-1, GSDMD, and IL-1β was reduced, but there was no significant difference in IHC analysis, which limited to the sample size (Fig. 5A and B).Nevertheless, the immunoblot analysis showed that the expression of NLRP3 and GSDMD were significantly reduced after treatment with DSF (Fig. 5C and D).For apoptotic signaling, the level of cleaved caspase-3 in the DSF-treated group was increased, which was more evident in the high-concentration DSF group (Fig. 5A-D).The isotype control and the negative control revealed a high specificity of the primary antibody used in mouse samples (Fig. S10).Overall, these results suggested that DSF ameliorates IMQ-mediated psoriatic lesions by suppressing pyroptotic progression and promoting the apoptotic signaling pathway.

Disulfiram might suppress the activation of M1 macrophages to reduce the proliferation of keratinocytes through EGF signaling
Eight target genes of DSF were identified from the L1000 database, including ALDH1A2, CYP2E1, ALDH2, DBH, DNMT1, ALDH5A1, TRPA1, and ALDH7A1.Based on these gene sets, the target gene enrichment fraction of DSF was significantly increased in M1 macrophages (Fig. 6A  and B).Previously, our histological analysis showed that DSF could decrease the thickness of the skin fold in the epidermis, which is mainly composed of keratinocytes.Thus, we analyzed the cellular communication between M1 macrophages and keratinocytes (Fig. 6C).Based on the cellular markers (Table S9) [45][46][47][48], three skin keratinocytes were identified in the UMAP diagram, including basal keratinocytes (BCs), spinous cells (SCs) and mitotic cells (Fig. S8A).Obviously, the epidermal growth factor (EGF) signaling pathway between M1 macrophages and keratinocytes was significantly upregulated in psoriatic patients (Fig. 6D  and E).
To further clarify the mechanism, we separately investigated the signal changes and expression changes in the EGF signaling pathway in macrophages and keratinocytes (Fig. 6F and G).The results suggested that the ligand proteins (EREG) were specifically highly expressed in M1 macrophages, while their receptor proteins (epidermal growth factor receptor (epiregulin (EGFR)) were specifically highly expressed in keratinocytes (including basal keratinocytes (BCs), spinous cells (SCs) and mitotic cells) (Fig. 6G).Next, we compared the probability of communication between M1 macrophages and three kinds of keratinocytes mediated by ligand-receptor pairs in the EGF signaling pathway (Fig. S12A and B).It is worth noting that the communication probability of the EREG-EGFR receptor ligand pair between M1 macrophages and keratinocytes increased significantly in psoriasis (Fig. S12A).

Discussion
Recent new insights into the pathogenesis of psoriasis have increased the attention given to inflammatory cell death, including NLRP3mediated pyroptosis, caspase-mediated apoptosis, and RIP-mediated necroptosis.PANoptosis serves as a newly emerging RCD that is extensively activated in complex crosstalk and coordination.However, the role of PANoptosis in psoriasis remains unexplored.By taking full advantage of RNA-Seq, scRNA-Seq and experimental analysis of human skin samples from patients with psoriasis, we identified a high activity of PANoptosis signaling in the psoriatic group.Furthermore, two distinct PANoptosis patterns were identified, and a significantly dysregulated immune response was found in the high PANoptosis-related pattern.Based on the hub genes from PANoptosis-mediated immune signaling, DSF was explored as a candidate drug through network pharmacology and experimental validation.Moreover, DSF suppressed pyroptosis signaling and enhanced apoptosis signaling, contributing to ameliorating psoriasis-like symptoms.These findings might provide valuable clues for a breakthrough into psoriasis therapies.
PANoptosis is a coordinated and systematic RCD that occurs among three of these RCDs (pyroptosis, apoptosis, and necroptosis) and is strongly related to inflammatory and immune responses [49][50][51].In this study, high activity of the PANoptosis pathway was identified in psoriatic lesions.Accumulating studies have separately revealed the role of these three RCDs in contributing to the development of psoriasis.For example, a previous study reported that NLRP3 inflammasome-mediated pyroptosis was present in IMQ-induced psoriasis, similar to skin inflammation in mice [52][53][54].Further study showed that TNF-α-mediated the NLRP3 inflammasome in psoriatic patients contributed to systemic inflammation [55].Unlike the explosive rupture and cytoplasm flattening in pyroptosis, apoptosis is generally believed to be a safe form of RCD [56,57].In psoriasis, evasion of keratinocyte apoptosis plays an important role in facilitating abnormal keratinocyte hyperproliferation [58,59].Subsequently, various potential therapeutic strategies for psoriasis have emerged to promote keratinocyte apoptosis, such as miR-383 [18], mesenchymal stem cells (MSCs) [32], and BAY 11-7082 antagonists [60] Based on the highlighted PANoptosis pathway in psoriasis, we identified two distinct PANoptosis patterns in patients with psoriasis.PANoptosis serves as a coordinated RCD signaling-enabled crosstalk and coregulation among these RCDs and is more appropriate in a complex microenvironment during the development of psoriasis.Upon further analysis of the PANoptosis-related classification, a series of inflammatory and immune signals were enriched in the high PANoptosis-related  pattern.As reported, an increased circulating level of cytokines (e.g., TNF and IFN-γ) could synergistically induce PANoptosis characterized by activating the molecules involved in pyroptosis (GSDMD), apoptosis (CASP8/3/7) and necroptosis (pMLKL) [5,62].These cytokines could further activate JAK/STAT1/IRF1 signaling and nitric oxide (NO) production to enhance CASP8/FADD-mediated PANoptosis [5].During PANoptosis, the excessive production of cytokines is mediated, known as cytokine storm (CS).Thus, PANoptosis might lead to a cytokine-cell death positive-feedback loop in the progression of psoriasis.This possibility and mechanism require further study.
Additionally, the majority of cell death studies have been performed with innate immune cells, such as macrophages.Mechanistically, ZBP1, characterized as a critical innate immune sensor, could modulate cell death in the form of PANoptosis through the ZBP1-PANoptosome [9].It has been found that ZBP1-mediated sensing of influenza A virus (IAV) proteins could activate the NLRP3 inflammasome, leading to the production of IL-1β and IL-18 in mouse macrophages during IAV infection [63].Since different triggers occur in patients with psoriasis (e.g., stress, streptococcal infection, drinking, smoking, drug exposure, etc.) [64], the complex messages delivered by PANoptosis might be optimal for skin lesions to initiate inflammatory and immune signals to handle this systemic response.
Focusing on the mechanism of PANoptosis, we aimed to determine the diverse functions of hub genes involved in PANoptosis for drug prediction.In our experimental data, the predicted drug (DSF) could significantly ameliorate IMQ-mediated psoriatic lesions.And, we also found that DSF treatment could reduce the cleavage of GSDMD in IMQmediated psoriatic lesions.Recently, it has been also reported that topically application of DSF could reduce the cleavage of GSDMD, caspase-1, and IL-1β, which alleviated IMQ-mediated psoriatic lesions [65].DSF was originally used as an anti-alcoholism drug by acting on aldehyde dehydrogenase (ALDH), which was approved by the FDA in 1951 [66].DSF has also been identified as an effective inhibitor of GSDMD pore formation against cellular pyroptotic progress, which suggests that DSF is a potential drug for various inflammatory diseases [41,67].It was found that DSF did not directly inhibit priming or GSDMD cleavage [41].In psoriasis, GSDMD-mediated pyroptosis was involved in the inflammatory responses in complex immune microenvironment [65].Intradermal injection of DSF every other day for 7 days might reduce the inflammatory factors (e.g., IL-1β and TNF-α [38,39]) and suppress inflammatory cell infiltrations in IMQ-mediated psoriatic lesions, which might reduce the activation of NLRP3 inflammasome, caspase-1 processing and then the cleavage of GSDMD in the repaired psoriatic immune microenvironment.Or if there is a key factor act like an 'OFF' switch to limit upstream in pyroptosis during a long-term treatment of DSF? How DSF acts during this chronic pathogenesis of psoriasis needs to be further studied.
Additionally, scRNA analysis showed that DSF might suppress EREG expression in M1 macrophages in psoriasis, which could further reduce the activation of proliferation signaling in keratinocytes through downregulation of EREG-EGFR cooperation.The EGF family is identified as the major growth factor for the proliferation of epidermis to stimulate the growth of keratinocytes, which is critical for the hyperproliferation of epidermal keratinocytes in psoriasis [68][69][70].However, it still needs to be further verified and explored the expression level of the hub genes in PANoptosis-mediated immune signaling in psoriasis, whether DSF ameliorated IMQ-mediated psoriatic lesions by the direct or/and indirect regulation of these hub genes, and how DSF reduced the expression of the EGF family.Furthermore, the mechanism of the effectiveness of predicted drugs needs further exploration.
To our knowledge, this is the first report of PANoptosis signaling in skin inflammatory disease.Future work should focus on more discoveries to uncover the underlying mechanism of the PANoptosis-mediated immune response in skin inflammatory diseases, such as psoriasis and rosacea.We also look forward to revealing the role of PANoptosisinducing sensors and PANoptosome components induced by a variety of pathogenic triggers, which is critical to leveraging our understanding of PANoptosis, contributing to a breakthrough in therapies and improving the outcomes of patients with psoriasis.
In summary, we highlighted the critical role of PANoptosi signaling in the development of psoriasis.We established a systematic PANoptosis-mediated immune network during psoriatic aggravations, which provides novel insight for drug exploration and DSF and was verified as an effective drug to ameliorate psoriasis-like symptoms by suppressing the pyroptosis-mediated inflammatory response and enhancing apoptosis signaling.However, the mechanism and clinical effect of DSF on psoriasis require further investigation.

Fig. 1 .
Fig. 1.PANoptosis signaling highlighted in psoriasis.(A) The PANoptosis-related genes are shown in a Venn diagram.The detailed gene names are listed in Table S2.(B) Volcano plots highlighting the significantly different PANoptosis-related key genes (P < 0.05) between lesional and nonlesional skin from psoriasis patients shown in Table S3.Red indicates the significantly upregulated gene group, while blue indicates the downregulated group.(C) GSEA highlighted the PANoptosis reaction signaling in psoriasis.(D) Sample dendrogram and trait heatmap showing the PANoptosis signaling correlation in psoriasis.(E) Difference analysis of the key PANoptosis-related members (mainly pyroptosis).Statistical differences were compared by t test.(F) GSVA highlighted the PANoptosis reaction signaling in psoriasis.Statistical differences were compared by t test.(G) Heatmap showing the expression of PANoptosis-related genes in each sample.The RNA-Seq data were obtained from the GEO database (GSE30999), which involved 85 nonlesional and 85 lesions of psoriasis.(H) IHC staining presented the part of PANoptosis-related key proteins (pyroptosis) in psoriasis (psoriasis = 8, healthy individual = 6).Scale bar: 20 µm.(I) Quick-score analysis of IHC.Statistical differences were compared by Mann-Whitney test (*** P < 0.001, NS, not statistically significant.).IHC, immunohistochemistry; RNA-Seq, RNA sequencing.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 1.PANoptosis signaling highlighted in psoriasis.(A) The PANoptosis-related genes are shown in a Venn diagram.The detailed gene names are listed in Table S2.(B) Volcano plots highlighting the significantly different PANoptosis-related key genes (P < 0.05) between lesional and nonlesional skin from psoriasis patients shown in Table S3.Red indicates the significantly upregulated gene group, while blue indicates the downregulated group.(C) GSEA highlighted the PANoptosis reaction signaling in psoriasis.(D) Sample dendrogram and trait heatmap showing the PANoptosis signaling correlation in psoriasis.(E) Difference analysis of the key PANoptosis-related members (mainly pyroptosis).Statistical differences were compared by t test.(F) GSVA highlighted the PANoptosis reaction signaling in psoriasis.Statistical differences were compared by t test.(G) Heatmap showing the expression of PANoptosis-related genes in each sample.The RNA-Seq data were obtained from the GEO database (GSE30999), which involved 85 nonlesional and 85 lesions of psoriasis.(H) IHC staining presented the part of PANoptosis-related key proteins (pyroptosis) in psoriasis (psoriasis = 8, healthy individual = 6).Scale bar: 20 µm.(I) Quick-score analysis of IHC.Statistical differences were compared by Mann-Whitney test (*** P < 0.001, NS, not statistically significant.).IHC, immunohistochemistry; RNA-Seq, RNA sequencing.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) X.-m.Hu et al.

Fig. 3 .
Fig. 3. Landscape of immune cell infiltration and potential drug prediction based on PANoptosis/immune signaling.(A) The relationships among modules, clinical characteristics, PANoptosis signaling and highlighted immune cell infiltration in psoriasis (red indicates a positive correlation, while blue indicates a negative correlation).(B) The relationship between GS and MM in the black modules (highest related module).Genes whose MM > 0.8 and GS > 0.2 were selected as candidates for drug prediction.(C) Drug prediction on the Enricher website based on the hub genes and significantly different genes involved in molecular patterns.Structure-based virtual screening of 4KA4 (ZBP1) was performed to select the candidate drugs more precisely (Table S7).(D) Molecular docking of DSF and ZBP1/ Caspase-1/CXCL1 (the red frame indicates the DSF structure).MM, module membership; GS, gene significance; DSF, disulfiram.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) . Necroptosis is a new type of programmed necrosis that can strongly facilitate the inflammatory response by inducing the manufacture of cytokines and disrupting damage-associated molecular pattern (DAMP) release [61].Duan et al. highlighted the proinflammatory effect of necroptosis contributing to the development of psoriasis, and necroptotic inhibitors (RIPK1 R-7-Cl-O-Necrostatin-1 (Nec-1 s) and MLKL-inhibitor necrosulfonamide (NSA)) could powerfully block IMQ-induced inflammatory responses [17].Combined with these previous findings and our discoveries, we highlighted the critical role of PANoptosis in the development of psoriasis.

Fig. 4 .Fig. 5 .
Fig. 4. DSF was effective against psoriasis as a candidate drug.(A) Diagram of animal modeling for DSF treatment in psoriasis (n = 4 for each group).(B) PASI analysis during the animal modeling.(C) The basic characteristics of mice are presented in phenotype and H&E staining.(D) The spleen phenotype is shown in each group.(E) Epidermal thickness was analyzed based on H&E staining.(F) The spleen index was tested based on the weight of the spleen and body.(G) EthD-III staining (red) was labeled as pyroptosis, and Hoechst (blue) staining was tagged as nuclei.(H) TUNEL staining (red) was labeled as apoptosis, and Hoechst (blue) staining was tagged as nuclei.Scale bar: 20 µm.(I) The positively labeled cells were analyzed for pyroptosis reaction evaluation (EthD-III staining).(J) The positively labeled cells were analyzed for apoptosis reaction evaluation (TUNEL staining).Statistical differences were compared by one-way ANOVA.(* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.NS, not statistically significant).DSF, disulfiram; H&E, hematoxylin and eosin; PASI, psoriasis area severity index.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.Upregulation of the EGF signaling pathway between M1 macrophages and keratinocytes in psoriasis.(A) Reduced-dimensional UMAP diagrams of 18 immune cell subsets formed by immune cell subgrouping; each color represents each immune cell subgroup, and each dot in the UMAP diagram represents a cell.(B) A twodimensional projection UMAP map of the targeting gene set of DSF scored in each cell.(C) Quantitative circle diagram of receptor ligand pairs between macrophages and keratinocytes in psoriasis.Each color represents a class of cells, and the thicker the connection between the two cells, the more communication there is.(D) Comparative heatmap of outgoing signals of various signaling pathways between the psoriasis and control groups.(E) Comparative heatmap of incoming signals of various signaling pathways between the psoriasis and control groups.(F) Comparative heatmap of overall signals of EGF signaling pathways between the psoriasis and control groups.(G) Expression of each molecule in the EGF signaling pathway in 6 kinds of cells.BC, basal keratinocyte; SC, spinous cell; EGF, epidermal growth factor; UMAP, uniform manifold approximation and projection; DSF, disulfiram.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)